Composition and method for the encapsulation of water-soluble molecules into nanoparticles

ABSTRACT

A method and composition for the encapsulation of hydrophilic molecules in submicron particles is disclosed. The particles are composed of a water-in-oil microemulsion surrounded by one or more biocompatible polymers.

BACKGROUND OF THE INVENTION

Encapsulation of drugs into microparticles (e.g. nanoparticle andnanocapsule delivery systems) provides several advantages for in vivodrug delivery, including the ability to modify the drug'sbiodistribution and to increase its bioavailability. These advantagesare particularly important for therapeutic polar (i.e., water-soluble)macromolecules, such as polypeptides, polysaccharides, andpolynucleotides, which otherwise have poor bioavailability, particularlywhen administered orally. However, prior to the present invention,satisfactory techniques for encapsulating water-soluble drugs intosubmicron particles were unknown.

One important feature of microparticles is the protection they afford todrugs from acid and enzymatic hydrolysis in the gut. For example, oncedelivered to a subject, submicron particles may be taken up via thegut-associated lymphoid tissue (GALT), commonly known as Peyer'spatches, into the lymphatic circulation. This route of uptake avoidshepatic first-pass metabolism and permits a therapeutic drug level to beachieved using a smaller dose, since metabolic systems need not besaturated. Moreover, intramuscularly or subcutaneously injectedsubmicron particles are also capable of entering the lymphatic systemand thus can circulate throughout the body. The material properties ofthe submicron particle wall or matrix also can be tailored to allowprogrammed release of the drug, thereby further improving the drug'sbiodistribution and bioavailability.

The polymers used to form the matrices or capsule walls ofmicroparticles are typically not water-soluble and therefore are notmiscible with water-soluble drugs. Accordingly, the water/oil/water(w/o/w) double emulsion process has typically been used to encapsulatehydrophilic drug molecules into microparticles. This process involvesthe dispersion of an aqueous solution containing drug into an organicphase containing a preformed polymer in solution. The primary water/oil(w/o) emulsion is in turn dispersed into a second aqueous phasecontaining an emulsion stabilizer. This technique has been shown toefficiently encapsulate hydrophilic drugs, such as proteins,polynucleotides, or polysaccharides, with adequate core loads intoparticles larger than 1 μm. However, particles greater than 1 μm aretaken up by the GALT far less efficiently than submicron particles.Therefore, the bioavailability of encapsulated molecules would besignificantly higher if particles containing high drug loads could bemanufactured in the submicron range.

Particle uptake via the GALT after oral delivery increases exponentiallyas particle size decreases from 5 μm into the submicron range.Similarly, in cases where it is desired that subcutaneously orintramuscularly injected particles circulate in tissues, particle sizemust be less than approximately 5 μm. However, the efficiency with whichdrug molecules can be encapsulated, particularly large, water-solublemolecules, decreases dramatically as particle diameter decreases belowapproximately 1 μm. Therefore, creating submicron particles capable ofbeing taken up efficiently by the GALT or capable of circulating withintissues that also contain sufficient drug content to allow therapeuticdrug concentrations to be achieved is one of the main challenges in thepharmaceutical industry.

In a typical encapsulation involving the coarse water/oil/water doubleemulsion technique, the internal aqueous phase is usually dispersed intooil at a volume ratio of 1:2 to 1:20 (w:o), with higher encapsulationefficiencies observed for lower ratios of water to oil. Particles assmall as 1 to 3 μm in diameter may be generated using this technique.However, the size of the internal water droplets has a lower limitdetermined by the physical properties of the internal water and oilphases. The size of the internal water droplets in turn determines theefficiency with which drug may be encapsulated in particles in thesubmicron size range.

In addition, another problem associated with past techniques of formingsubmicron particles is that coarse emulsions typically formed toencapsulate water-soluble molecules in microparticles are notthermodynamically stable. Internal water droplets will tend to fuse andbecome larger if the particles are not quickly hardened. As one attemptsto reduce the overall particle size, for example, by increasing mixingenergy, and/or decreasing the viscosity of the primary emulsion, theencapsulation efficiency decreases due to increased opportunity forinternal water droplets to diffuse to the outer surface of the oil phaseand deposit the contents of the internal aqueous phase into the externalaqueous medium. The end result of this thermodynamic instability of theinternal w/o emulsion is that the proportion of drug associated withpolymer becomes increasingly restricted to the surface of the particleswhich causes it to be quickly released (referred to as a “burst”) fromthe nanoparticle after dosing, a result which is often contrary to theintended release profile. In the case of oral delivery in particular,significant quantities of drug may be released before the particles aretaken up across the gut mucosa.

Other strategies have been attempted to efficiently encapsulatewater-soluble molecules into submicron particles. For example, naturallyoccurring hydrophilic polymers, such as albumin or gelatin, have beenused to generate matrix-type nanoparticles. However, while hydrophilicpolymers are compatible with water-soluble drugs and therefore have thepotential for high loads and high encapsulation efficiencies, thehydrophilic surfaces of these particles are less likely to be taken upvia the GALT than are particles of similar size with hydrophobicsurfaces. Moreover, extensive processing is often required to removetoxic chemical crosslinking agents used to harden the particles. Heatdenaturation has also been used to form hardened particles with theproblem that heat often destroys the bioactivity of encapsulated drugs.

Methods for encapsulating drugs into microparticles composed ofpreformed polymers, such as the spontaneous emulsification process, havealso been described (see e.g., U.S. Pat. No. 5,118,528). However, whilesubmicron particles with uniform size distributions have been formedusing this technique, it has been shown that large, water-soluble drugsare not efficiently encapsulated and high burst release characteristicsare common (Niwa et al. (1994) J. Pharm. Sci. 83:727). Another drawbackto the technique is that only limited volumes of aqueous drug solutionscan be added to the polymer solution without affecting polymersolubility when hydrophobic polymers are used. Furthermore, lowmolecular weight polymers with a higher polar character than polylacticacid tend to precipitate without encapsulating, rather than formnanoparticles.

U.S. Pat. No. 5,049,322 describes a modified technique for theproduction of nanocapsules using preformed polymers. In this technique,an oil, a solid suspension, or volatile organic solution containing drugis dispersed into a water-miscible organic solvent, usually acetone,containing a solution of polymer. A polymer wall is deposited aroundsolid particles or oil droplets when the oil phase is poured into asecond continuous, usually aqueous phase that is a nonsolvent for thepolymer. However, this patent describes a system for the encapsulationof material compatible with oils or organic solvents rather than aqueoussolutions.

Other nanoparticle encapsulation techniques, such as the phase inversionmethod described in U.S. Pat. No. 6,143,211, require that thehydrophilic drug molecule be in a micronized, solid form and besuspended, rather than dissolved, in the organic phase. This has thedisadvantage that dehydration of certain classes of water-solublemolecules, such as proteins, in addition to requiring expensive materialprocessing steps, often results in irreversible structural andfunctional damage. Furthermore, dissolution of encapsulated material intissue fluids may be incomplete, or, in the case of proteins, may formimmunogenic aggregates.

Accordingly, improved techniques for efficiently encapsulatinghydrophilic (water-soluble) drugs into microparticles, particularlysubmicron particles, at core loads that result in pharmacologicalactivity, particularly after oral delivery, would be of great benefit.

SUMMARY OF THE INVENTION

The present invention provides an improved method and composition forencapsulating a wide variety of water-soluble agents intomicroparticles, including submicron particles (e.g., nanoparticles orparticles having a size of less than about 1000 nanometers), capable ofeffectively delivering such agents in vivo, particularly whenadministered to subjects orally. The method involves forming amicroemulsion containing an aqueous drug solution solubilized in oil,and subsequently encapsulating the microemulsion in a polymer shell. Theresulting microparticles contain high drug core loads with minimalsurface adsorbed drug, thereby reducing any burst effect.

While the method of the present invention has particular advantages forthe encapsulation of water-soluble molecules into submicron particles,the method also can be used to form microparticles of larger size. Thismay be preferable, for example, in instances where delivery ofwater-soluble agents from a non-circulating injected depot is desired.

Accordingly, in one embodiment, the present invention provides a methodfor encapsulating a water-soluble agent by (a) forming a microemulsioncomprising the agent; (b) adding the microemulsion to a first solventcomprising one or more polymers, thereby forming a dispersion; and then(c) adding the dispersion to a second solvent which is a nonsolvent forone or more polymers, resulting in encapsulation of the microemulsion bythe one or more polymers in the form of microparticles.

In a particular embodiment, the drug-containing microemulsion formed inthe invention comprises about 10% to 60% oil by volume. Themicroemulsion can further contain one or more surfactants orco-surfactants. Suitable surfactants include, but are not limited topolyoxyethylene sorbitan monooleate alone, sorbitan monolaurate, andmixtures thereof. Suitable co-surfactants include but are not limited toshort to medium chain alkyl- or branched chain alcohols, such asethanol, propanol, isopropanol, butanol, isobutanol, pentanol andisopentanol.

Typically, the microemulsion is added to the first solvent containing apolymer at a concentration of about 0.01% to 30% (w/w), more preferablyabout 0.1% to 10% (w/w). Suitable polymers include, for example, organicpolymers such as polyvinyl alcohols, polyvinyl ethers, polyamides,polyvinyl esters, polyvinylpyrrolidone, polyglycolides, polyurethanes,alkyl celluloses, cellulose esters, hydroxypropyl derivatives ofcelluloses and cellulose esters, preformed polymers of poly alkylacrylates, polyethylene, polystyrene, polyactic acid, polyglycolic acid,poly(lactide-co-glycolide), polycaprolactones, polybutyric acids,polyvaleric acid and copolymers thereof, alginates, chitosans, gelatin,albumin, zein and combinations thereof. A preferred polymer ispoly(lactide-co-glycolide). Accordingly, the first solvent can be anysuitable solvent for such polymers including, for example, ethylacetate, benzyl alcohol and propylene carbonate.

In contrast to the first solvent, the second solvent is a nonsolvent forthe selected polymer(s). A preferred second solvent is water. The secondsolvent can further include one or more emulsifying agents orsurfactants to improve the formation of the microparticles upon additionof the microemulsion-containing dispersion.

Microparticles are then formed by adding the dispersion to the secondsolvent. The microparticles are typically in the submicron size rangeand are composed of a microemulsion containing a water-soluble agentencapsulated by one or more polymers.

A wide variety of water-soluble therapeutic agents, such as proteins,peptides, nucleic acids and other polar drugs, can be encapsulated usingthe method and composition of the present invention. Moreover, theresulting microparticles can be effectively administered to subjectsusing a variety of techniques, including parental (e.g., injection),topical and oral administration. Thus, the present invention is broadlyapplicable to many in vivo drug therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows heparin-containing nanoparticles prepared in accordancewith the present invention.

FIG. 2 is a graph showing the in vitro release rate of heparin fromnanoparticles prepared in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved methods and compositions fordrug delivery by enabling the encapsulation of water-soluble agents(e.g., drugs) by polymers in the form of microparticles, includingsubmicron particles. This is achieved by forming a microemulsioncontaining the agent, followed by encapsulation of the microemulsion.

I. Microemulsion Formation

As used herein, the term “microparticle” is defined generally asparticles that are less than about 1000 micrometers (μm) in size (e.g.are about 800 μm, 600 μm, 400 μm, 200 μm or less in size).Microparticles also include submicron particles, which are typicallyless than about 1000 nanometers (nm) in size (e.g. are about 800 mn-200mn, more preferably about 600 nm-200 nm or less in size) and which arecapable of effectively delivering water-soluble agents in vivo.

As used herein, the term “microemulsion” refers to a system of water,oil, and amphiphile which is a single optically isotropic andthermodynamically stable liquid solution (Danielsson and Lindman (1981)Colloids Surfaces 3:391-392). The term “microemulsion” does not imply orrequire any particular microstructure (e.g., involving a definiteboundary between oil and water phases). In addition, for purposes of thepresent invention, a microemulsion includes systems that areco-solvents, i.e. systems in which components may be molecularlydispersed. For example, the microemulsion may be a strict water in oilmicroemulsion, a bicontinuous monophase, a micellar solution, or swollenmicellar solution.

As used herein, the term “agent” refers to any water-soluble soluteincluding, but not limited to, proteins, peptides, polysaccharides,nucleic acids or other biologically active compounds for administrationto a subject, such as a human, animal or other mammal. While selectedbased upon the intended application or therapy, the agent is typically atherapeutic water-soluble drug, the efficacy of which can be improved oroptimized when administered orally with a programmable, extended releasepharmacokinetic profile, as is accorded by the present invention.

Suitable therapeutic proteins for use in the present invention include,for example, interferon-alphas, interferon-betas, interferon-y,erythropoetins, granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins,asparaginase, adenosine deaminase and insulin, to name but a few.

Suitable therapeutic peptides for use in the present invention include,for example, hormones such as adrenocorticotropin (ACTH), glucagon,somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentaryhormones, somatomedin, lutenizing hormone, chorionic gonadotropin,hypothalmic releasing factors, antidiuretic hormones, thyroidstimulating hormone, endorphins, enkephalins, biphalin and prolactin.

Additional suitable proteins include monoclonal and polyclonalantibodies, single-chain antibodies, other antibody fragments, analogsand derivatives thereof. Polynucleotides, including antisenseoligonucleotides, aptamers and therapeutic genes can also be deliveredusing the methods and compositions of the present invention.

Anticoagulants, such as heparin and low molecular weight heparin, alsocan be delivered using the methods and compositions of the invention.Still other suitable therapeutic agents for use in the present inventioninclude small bioactive molecules, such as anticancer drugs, e.g.,doxorubicin and daunorubicin, vincristine, cisplatin, carboplatin,camptothecin and camptothecin analogs, antibiotics, antipsychotics,antidepressants, and small molecule drugs for diabetes andcardiovascular disease.

Typically, the molecular weight of the encapsulated water-soluble agentused in the present invention is about 10,000,000 daltons or less.Specific examples include for example, but are not limited to, eosin at624 daltons, heparin at 25,000 daltons and DNA plasmids at about10,000,000 daltons. However, incorporation of a water-soluble agent intoa microemulsion should not be limited by molecular weight.

Many thermodynamically stable water-oil-surfactant-co-surfactantmicroemulsion systems exist that can solubilize significant quantitiesof water or aqueous solutions, and thus can be used in the presentinvention. Preferred microemulsion components are orally or parenterallybiocompatible. For example, suitable oils for use in forming themicroemulsion include, but are not limited to, vegetable oils, purifiedsynthetic or natural triglycerides, phospholipids and their derivatives,such as lecithin or lysolecithin, alone or in mixtures with other oils.These oils are preferred to hydrocarbons, although microemulsionsemploying hydrocarbon oils, such as the n-decane through n-octadecaneseries, are also within the scope of the invention.

Other organic liquids including, but not limited to, benzene,tetrahydrofuran, and n-methyl pyrrolidone, or halogenated hydrocarbons,such as methylene chloride, or chloroform may also be used as the oilcomponent of the microemulsion. In a particularly preferred embodimentof the invention, fatty acid esters, for example, isopropyl myristate orethyl oleate, are used as the oil phase of the microemulsion. Each oilor unique mixture of oils may require a different surfactant or mixtureof surfactants or surfactants and co-surfactants to solubilize water, ascan routinely be determined by those of skill in the art. The proportionof oil or mixture of oils used in the microemulsion is typically in therange of between 10 and 60% by volume, most preferably between 20 and50% by volume.

In general, the microemulsion also includes one or more surfactants orco-surfactants, as is well known in the art. Any surfactant may be usedwhich, alone, or in combination with a co-surfactant, reduces theinterfacial tension between oil and water components sufficiently (e.g.,<10⁻³ dyn/cm) to allow the spontaneous formation of a water in oilmicroemulsion. Examples include, but are not limited to, anionicsurfactants such as fatty acid soaps, acyl sulfates, or acylsulfosuccinates; cationic surfactants, such as alkyl primary, secondary,tertiary, or quaternary amines; nonionic surfactants, for example,sorbitan esters or polyethoxylated esters of acyl acids, copolymers ofpolyethylene oxide and polypropylene oxide. In a preferred embodiment ofthe invention, biocompatible surfactants, such as polyoxyethylenesorbitan monooleate, alone or in a mixture with sorbitan monolaurate areused to solubilize water in oil. The content of the surfactant ormixture of surfactants, apart from alcohols or other co-surfactants inthe microemulsion, can range for example from between 0.1 to 60% byvolume, more preferably from between 10 to 50%.

In accordance with the present invention, co-surfactants may also beused in microemulsion systems to increase interfacial pressure betweenwater and oil phases. Microemulsion stability depends, in part, on theinteraction between carbon chains of the oil, surfactant, andco-surfactant (BSO theory). Thus, for a given oil-surfactant system,water solubilization can be varied as the alcohol co-surfactant chainlength is varied, as is well known in the art. In a preferredembodiment, the co-surfactant is an aliphatic alcohol, more preferably aprimary aliphatic alcohol. Shorter chain alcohols, such as ethanol, areparticularly preferred to more toxic, longer chain alcohols for use asco-surfactants. Alcohol content may range, for example, from about 0 toabout 30% by volume in the microemulsion, more preferably from about 5to about 20%.

Microemulsion formation from the above component categories proceedsspontaneously due to the favorable free energy of formation as thecomponents are mixed together. Although microemulsions arethermodynamically favored, kinetic barriers may in some instances impedetheir formation. Accordingly, the application of moderate increases inmixing energy or temperature can be applied if necessary to overcomesuch kinetic barriers to the formation of the microemulsion, as is wellknown in the art.

II. Dispersion Formation

Following formation of the microemulsion containing the water-solubleagent, the microemulsion is added to a solvent (e.g., an organicsolvent) containing one or more polymers, thereby forming a dispersion.

As used herein, the term “dispersion” refers to the distribution ofparticles throughout a medium, such as a solvent. The term “solvent”refers to any liquid substance that is capable of dissolving,dispersing, or suspending one or more other substances. Accordingly,suitable first solvents for use in the present invention include anysolvent or mixture of solvents in which the polymer is soluble and inwhich the microemulsion can be dispersed. For example, suitable polymersolvents (i.e., for use as the first solvent) include, but are notlimited to ethyl acetate, propylene carbonate, benzyl alcohol,acetonitrile and other organic solvents which are completely orpartially miscible with the second solvent used in the invention.

As used herein, the term “polymer” includes any film forming polymer ofnatural, synthetic, or semi synthetic origin, and may be biodegradableor nonbiodegradable. Examples of suitable polymers include, but are notlimited to, polyvinyl alcohols, polyvinyl ethers, polyamides, polyvinylesters, polyvinylpyrrolidone, polyglycolides, polyurethanes, alkylcelluloses, cellulose esters, hydroxypropyl derivatives of cellulosesand cellulose esters, preformed polymers of polyalkyl acrylates,polyethylene, polystyrene, polylactic acid, polyglycolic acid,polycaprolactones, polybutyric acid, polyvaleric acid and copolymersthereof, alginates, chitosans, gelatin, albumin, zein, alone, asphysical mixtures, or as copolymers.

In a preferred embodiment, polyesters, such aspoly(lactide-co-glycolide), are used to encapsulate the microemulsion.Such polyesters are typically in the molecular weight range of about1,000 to 250,000 daltons, preferably from about 3,000 to 150,000daltons. Moreover, the release rate of the resulting microparticles canbe varied by the molecular weight and concentration of the polymer. Thisconcentration typically ranges from about 0.01 to 30% (w/w), preferablyfrom about 0.1 to 10% (W/W).

III. Microparticle Formation

Following addition of the microemulsion to the polymer-containingsolvent (the first solvent), the resulting dispersion is then added to asecond solvent which is a nonsolvent for the polymer, thereby formingmicroparticles which encapsulate the microemulsion.

As used herein, the term “nonsolvent” refers to a medium (e.g., liquid)in which a given compound is not soluble (e.g., capable of beingdissolved and/or dispersed). For example, water is a nonsolvent forcertain organic polymers which are not soluble in water. Accordingly,any solvent that is a nonsolvent for the polymer(s) employed in thepresent invention can be used as the second solvent.

The second solvent employed in the present invention can be completelyor partially miscible with the first solvent, or non-miscible with thefirst solvent. Accordingly, in a particular embodiment, the secondsolvent is completely miscible with the first solvent. For example, thesecond solvent can be water and the first solvent can be acetone. Inanother particular embodiment of the invention, the second solvent isessentially completely nonmiscible with the first solvent. For example,the second solvent can be water and the first solvent can be chloroform.

In another particular embodiment of the invention, the first solvent ispartially miscible in the second solvent so that the first solvent maybe completely extracted from the discontinuous phase of the emulsion bythe addition of a further quantity of the second solvent, or anothersolvent that is a nonsolvent for the polymer, and that will make up acontinuous, external phase of sufficient quantity such that the firstsolvent is fully miscible upon addition of the further quantity of thesecond solvent, or mixture of polymer nonsolvents. For example, in thisembodiment, the first solvent can be ethyl acetate, benzyl alcohol orpropylene carbonate, and the second solvent can be water.

Emulsion stabilizers can also be added to the second solvent prior toaddition of the dispersion-containing (internal phase-containing) firstsolvent. Suitable emulsifying agents are well known in the art and caninclude, for example, naturally occurring, synthetic, ionic, or nonionicemulsifying agents, such as polyvinyl alcohol and block copolymers ofpolyethylene oxide and polypropylene oxide. The concentration ofemulsifying agent in the second solvent (the continuous phase) typicallyranges from about 0 to 10% (w/v), preferably from about 0 to 5% (w/v).

Emulsification and polymer disposition may be effected using variousprocesses, depending on the miscibility of the desired solvents. Forexample, when the second solvent is completely miscible with the firstsolvent, emulsification and polymer deposition proceed spontaneouslywith gentle mixing at the large interfacial area formed when the firstsolvent is added to the second solvent (see, e.g U.S. Pat. Nos.5,118,528 and 5,049,322).

Alternatively, when the first solvent is only partially miscible withthe second solvent, or where the second solvent is nonmiscible with thefirst solvent, higher mixing energies are typically required to formdroplets (microparticles) of the desired size. Moreover, polymerdeposition at the interface of the second solvent and the first solventmay be effected in the case of partially miscible solvents by adding asufficient quantity of second solvent to completely solubilize the firstsolvent. In the case of immiscible solvents (i.e. incapable of mixing orattaining homogeneity), physical means, such as vacuum distillation, maybe used to remove the first solvent from the emulsion and cause polymerdeposition (see, e.g. U.S. Pat. No. 4,177,177).

Accordingly, using the foregoing techniques and components,microparticles which encapsulate water-soluble compounds dissolved in amicroemulsion are formed. The microparticles have the advantage of beingsmall in size, for example, ranging from approximately 50 nm to 5 μm,more typically from about 200 nm to 600 nm, yet retain high drug loadefficiencies of approximately 80%, and more typically 90% or greater.However, the emulsification conditions employed in the invention may beadjusted in manners well known to those of ordinary skill in the art toincrease particle size in any range up to, for example, 1000 μm whileretaining high encapsulation efficiencies.

Other features, advantages and embodiments of the invention will beapparent from the following examples which are meant to be illustrativeand, therefore, not limiting in any way.

EXAMPLES A. Example 1

Encapsulation of a Water-Soluble Dye Dissolved in a MicroemulsionSystem.

A water/oil microemulsion was prepared as follows: 1.0 ml isopropylmyristate was added to 1.4 ml of a surfactant mixture consisting ofpolyoxyethylene sorbitan monooleate (Tween 80): sorbitan monolaurate(Span 20): ethanol in a 45:30:25 volume ratio. Next, 0.6 ml of a 6%(w/v) eosin Y solution was added and the mixture briefly vortexed untilan optically clear, single phase solution resulted (it was confirmedthat eosin was not soluble in the oil or the ethyl acetate prior toformulation of the microemulsion nanocapsules).

Next, 1.0 ml of the microemulsion was dispersed into 10 ml ofwater-saturated ethyl acetate containing 250 mgpoly(lactide-co-glycolide) in a 50:50 mole ratio (PLGA 5050 DL2 low,Medisorb, Cincinnati, Ohio) by using a Powergen 125 laboratoryhomogenizer (Fisher Scientific, Pittsburgh, Pa.) at low speed. Themixture was then homogenized for two minutes followed by the addition of20 ml of ethyl acetate-saturated water containing 1% (w/v) polyvinylalcohol. The mixture was further homogenized for an additional 5 min. toform a coarse oil/water emulsion. This emulsion was poured slowly into200 ml of distilled water while stirring with a magnetic stir bar.Stirring was continued overnight at room temperature and ambientpressure to allow evaporation of the organic solvent. Unencapsulatedeosin was spectrophotometrically measured in the filtrate afterfiltering the nanoparticle suspension through a 0.02 micrometer porediameter membrane (Whatman Anodisk).

The resulting nanoparticles were found to contain 89.2% of the initialeosin, which represents a 2.9% eosin content by weight. Scanningelectron micrographs indicated that the particles ranged from 200 to 600nm in diameter (FIG. 1).

B. Example 2

Variation of Example 1

A water/oil microemulsion was prepared by adding 0.5 ml hexadecane to asurfactant mixture consisting of polyoxyethylene sorbitan monooleate(Tween 80): sorbitan monolaurate (Span 20): ethanol in a 45:30:25 volumeratio. To that mixture, 0.35 ml of a 6% (w/v) eosin Y solution wasadded. Eosin was found not to be soluble in hexadecane under theseconditions. Nanoparticles were formed and the eosin encapsulation wasmeasured as described in Example 1. The amount of eosin incorporatedinto nanocapsules was 98% of the initial quantity added. The particlesize distribution measured in electron micrographs was similar to thatfound in Example 1.

C. Example 3

Encapsulation of Heparin into Nanospheres Using Conventional SingleEmulsion Techniques

For purposes of comparing the drug loading (encapsulation) efficiency ofmicroparticles formed using the microemulsion technique of the presentinvention (e.g., as described in Examples 4 and 5 below), microparticlesformed using prior art techniques (e.g., the technique described in U.S.Pat. No. 5,049,322) were formed as follows. This technique was used tocompare drug loading levels with the present invention because thepolymer solvent is miscible with water and will allow the admixture ofaqueous heparin solutions, whereas other related nanoparticle formationtechniques are not compatible with the incorporation of aqueous drugsolutions.

A solution was prepared containing 12,110 USP units of heparin in 0.5 mldistilled water. This solution was admixed with a polymer solutionconsisting of 120 mg poly(lactide-co-glycolide) 50:50 mole ratio(Medisorb PLGA 5050 DL low), 15.0 ml acetone, 0.5 ml methylene chloride,and 1.0 ml water. The mixture formed a clear, single phase. The solutionwas poured into 50 ml of water containing 250 mg polyvinyl alcohol withmoderate stirring (magnetic stir bar rotating at approximately 100 rpm).A bluish opalescent suspension immediately formed. The suspension wasstirred overnight at ambient temperature and pressure to allow theorganic solvents to evaporate.

A portion of the suspension was next separated from the surroundingliquid by filtration through a 0.02 micrometer pore membrane. The solidswere washed with distilled water and solubilized with dimethyl sulfoxide(DMSO). The DMSO solution was diluted with a solution of 0.1% sodiumdodecyl sulfate (SDS) in 50 mM aqueous sodium hydroxide. Aliquots wereassayed for heparin activity using the anti-factor Xa enzyme assay(Sigma). Encapsulation efficiency was determined by comparing theheparin content of the washed nanoparticle suspension to the heparincontent of an equivalent volume of the unfractionated suspension thatwas solubilized with DMSO and diluted with the alkaline SDS solution.The encapsulation efficiency was calculated to be the ratio between theheparin content of the washed nanoparticles and the whole suspension,and was found to be 0.5%. Heparin content was 0.01% by weight.

D. Example 4

Encapsulation of a Microemulsion Containing Heparin

A water/oil microemulsion was prepared by adding 2.5 ml isopropylmyristate to 3.65 ml of a surfactant mixture consisting ofpolyoxyethylene sorbitan monooleate (Tweon 80): sorbitan monolaurate(Span 20): ethanol in a 45:30:25 volume ratio. The oil and surfactantwere blended together using a laboratory benchtop vortexer. Water, 1.45ml, containing 25,000 USP units of heparin was subsequently mixed withthe oil/surfactant blend by brief vortexing to form the microemulsion.

A 1.0 ml aliquot of the microemulsion containing heparin was then addedto 10.0 ml of water-saturated ethyl acetate containing 250 mgpoly(lactide-co-glycolide) in a 50:50 mole ratio (PLGA 5050 (Medisorb,Cincinnati, Ohio)). The mixture was homogenized for ninety seconds,added to 20 ml of ethyl acetate-saturated water containing 5% (w/v)polyvinyl alcohol and then further homogenized for 5 min. until a coarseoil/water emulsion formed. This emulsion was next poured slowly into 200ml of distilled water while stirring with a magnetic stir bar. Stirringwas allowed to continue overnight at room temperature and ambientpressure to facilitate evaporation of the organic solvent.

The following morning, a 10 ml aliquot of the nanoparticle suspensionwas added to a dialysis bag composed of cellulose ester with a nominalmolecular weight cutoff of 300 kD. The bag was dialyzed to equilibriumagainst distilled water. Heparin activity in the water was measuredusing a commercial anti-factor Xa enzyme assay (Sigma, St. Louis, Mo.).The quantity of heparin measured in the dialysate represented 0.1% ofthe total quantity of heparin added, thus providing an encapsulationefficiency of 99.9%. Heparin loading was 1.7% by weight.

Accordingly, the results showed that the heparin content ofnanoparticles made using the microemulsion encapsulation technique was170-fold higher than that of matrix-type nanoparticles made using thespontaneous emulsification method detailed in Example 3.

E. Example 5

Variation of Example 4

A water/oil microemulsion was prepared by adding 1.5 ml ethyl oleate to2.625 ml of a surfactant mixture consisting of polyoxyethylene sorbitanmonooleate (Tween 80): sorbitan monolaurate (Span 20): ethanol in a45:30:25 volume ratio. The oil and surfactant were blended togetherusing a laboratory benchtop vortexer. Water, 0.975 ml, containing 16,870USP units of heparin was subsequently mixed with the oil/surfactantblend by brief vortexing to form the microemulsion.

A 1.0 ml aliquot of the microemulsion containing heparin was added to10.0 ml of water-saturated ethyl acetate containing 250 mgpoly(lactide-co-glycolide) in a 50:50 mole ratio (PLGA 5050 (Medisorb,Cincinnati, Ohio)). The mixture was homogenized for ninety seconds, thenadded to 20 ml of ethyl acetate-saturated water containing 1% (w/v)poloxamer 188 (Pluronic F68) and further homogenized for 5 min. forminga coarse oil/water emulsion. This emulsion was poured slowly into 200 mlof distilled water while stirring with a magnetic stir bar. Stirring wasallowed to continue overnight at room temperature and ambient pressureto facilitate the evaporation of the organic solvent. Heparinencapsulation was measured by the dialysis method described in example3. The encapsulation efficiency was 91% and the heparin loading was 2.6%by weight.

F. Example 6

In Vitro Release of Heparin from Nanoparticles Containing Microemulsion

A microemulsion was produced consisting of 2.5 ml isopropyl myristate,4.7 ml of a Tween 80:Span 20:Ethanol (45:20:35) surfactant mixture, and1.75 ml water containing 300 mg heparin. Next, a 1.5 ml aliquot of thismicroemulsion was dispersed into 15 ml ethyl acetate containing 375 mgPLGA (50:50 L:G, mw 17,000 Daltons, Medisorb, Cincinnati, Ohio) using aliquid shear homogenizer for 1.5 min. While continuing homogenization,the dispersion was slowly poured into 30 ml aqueous 1% (w/v) polyvinylalcohol which had been presaturated with ethyl acetate. Homogenizationwas continued for an additional five minutes before pouring theresulting emulsion into 200 ml of distilled water while stirring with amagnetic bar. Stirring was continued overnight to allow evaporation ofthe organic solvent before the removal of unencapsulated heparin fromthe nanoparticles by gel filtration. A quantity of mannitol, 2.5 g, wasadded to the cleaned nanoparticle suspension prior to freeze-drying tofacilitate handling of the dried particles.

Approximately one gram of the dried nanoparticle formulation was addedto 100 ml phosphate buffered saline containing 25% ethanol, covered, andincubated at 37° C. One ml of the particle suspension was removed atprescribed intervals and centrifuged at 20,000× g for 30 min.Supernatants were collected and the heparin content was measured using acommercial anti factor Xa calorimetric assay. The in vitro releaseprofile is shown in FIG. 2.

G. Example 7

In Vivo Release of Heparin from Nanoparticles Containing Microemulsion

A microemulsion was produced consisting of 1.8 ml isopropyl myristate,2.7 ml of a Tween 80:Span 20:Ethanol (45:30:25) surfactant mixture, and0.9 ml water containing 135 mg heparin. One ml of this microemulsion wasdispersed into 10 ml of a 2.5% (w/v) PLGA solution in water-saturatedethyl acetate. The dispersion was mixed using a liquid shear homogenizerfor 1.5 min. Next, the dispersion was slowly poured into 20 ml ethylacetate-saturated water containing 0.1% (w/v) polyvinyl alcohol, andhomogenized under the same conditions for an additional 5 minutes. Anadditional 170 ml of distilled water was slowly poured into theemulsion. The vessel was stirred on a magnetic stir plate at roomtemperature overnight to allow evaporation of the organic solvent.

After evaporation, an aliquot of the suspension was filtered through a0.02 micron filter, and the heparin content of the filtrate was measuredusing a calorimetric anti-factor Xa enzyme assay. Encapsulated heparinwas found to be 94% of the total amount of drug added at the beginningof the encapsulation process. The formulation was freeze-dried andresuspended in a small volume of water to a concentration of 812 Unitsheparin per ml, and injected subcutaneously into male Sprague Dawleyrats weighing approximately 300 grams. One ml of blood was drawn fromthe tail vein prior to nanocapsule injection and at intervalsthereafter. Plasma was separated and assayed for heparin content using acolorimetric anti factor Xa enzyme assay. Table I shows heparin bloodlevels in Units/ml plasma at each time point.

Table I Heparin Concentration in Rat Plasma After SubcutaneousInjection.

TABLE I Heparin concentration in rat plasma after subcutaneousinjection. Time (hours) Animal 0 24 48 72 1 0.06 0.08 0.15 0.09 2 0.080.09 0.12 0.1  3 0.07 0.07 0.12 0.05 Mean 0.07 0.08 0.13 0.08 SD 0.010.01 0.02 0.02 The increase in plasma heparin noted at 48 hours postinjection is significant at P ≧ 0.05.

The increase in plasma heparin noted at 48 hours post injection issignificant at P≧0.05.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. In addition, theentire contents of all patents and publications cited herein are herebyincorporated by reference.

I claim:
 1. A method for encapsulating a water-soluble agent comprising:(a) forming a microemulsion containing the agent; (b) adding themicroemulsion to a first solvent comprising one or more polymers,thereby forming a dispersion; (c) adding the dispersion to a secondsolvent which is a nonsolvent for one or more polymers; whereinfollowing step (c), the microemulsion is encapsulated by the one or morepolymers in the form of microparticles.
 2. The method of claim 1,wherein the first solvent is completely miscible with the secondsolvent.
 3. The method of claim 1, wherein the first solvent ispartially miscible with the second solvent.
 4. The method of claim 1,wherein the agent is a water-soluble drug.
 5. The method of claim 1,wherein the microemulsion comprises about 10% to 60% oil by volume. 6.The method of claim 1, wherein the microemulsion further comprises asurfactant.
 7. The method of claim 6, wherein the surfactant is selectedfrom the group consisting of polyoxyethylene sorbitan monoleate alone,sorbitan monolaurate, and mixtures thereof.
 8. The method of claim 7,wherein the microemulsion comprises about 0.1% and 60% surfactant byvolume.
 9. The method of claim 1, wherein the microemulsion furthercomprises a co-surfactant.
 10. The method of claim 1, wherein thepolymer is selected from the group consisting of polyvinyl alcohols,polyvinyl ethers, polyamides, polyvinyl esters, polyvinylpyrrolidone,polyglycolides, polyurethanes, alkyl celluloses, cellulose esters,hydroxypropyl derivatives of celluloses and cellulose esters, preformedpolymers of poly alkyl acrylates, polyethylene, polystyrene, polyacticacid, polyglycolic acid, poly(lactide-co-glycolide), polycaprolactones,polybutyric acids, polyvaleric acid and copolymers thereof, alginates,chitosans, gelatin, albumin, zein and combinations thereof.
 11. Themethod of claim 10, wherein the polymer is poly(lactide-co-glycolide).12. The method of claim 1, wherein the polymer has a molecular weight inthe range of 1000 daltons to 150,000 daltons.
 13. The method of claim12, wherein the polymer has a molecular weight in the range of 3000daltons to 150,000 daltons.
 14. The method of claim 1, wherein thepolymer is present at a concentration of about 0.01% to 30% (w/w). 15.The method of claim 14, wherein the polymer is present at aconcentration of about 0.01% to 10% (w/w).
 16. The method of claim 1,wherein the first solvent is an organic solvent.
 17. The method of claim1, wherein the first solvent is selected from the group consisting ofethyl acetate, benzyl alcohol, and propylene carbonate.
 18. The methodof claim 1, wherein the second solvent is water.
 19. The method of claim1, further comprising the step of adding a second solvent prior to theaddition of the dispersion.
 20. A microparticle composition prepared bythe process of claim
 1. 21. A microparticle composition comprising amicroemulsion containing a water-soluble agent and one or more polymers.22. The composition of claim 21, wherein the polymer encapsulates themicroemulsion.
 23. The composition of claim 21, wherein the agent is awater-soluble drug.
 24. The composition of claim 21, wherein themicroemulsion comprises about 10% to 60% oil by volume.
 25. Thecomposition of claim 21, wherein the microemulsion further comprises asurfactant.
 26. The composition of claim 25, wherein the surfactant isselected from the group consisting of polyoxyethylene sorbitan monoleatealone, sorbitan monolaurate, and mixtures thereof.
 27. The compositionof claim 26, wherein the microemulsion comprises about 0.1% and 60%surfactant by volume.
 28. The composition of claim 21, wherein themicroemulsion further comprises a co-surfactant.
 29. The composition ofclaim 21, wherein the polymer is selected from the group consisting ofpolyvinyl alcohols, polyvinyl ethers, polyamides, polyvinyl esters,polyvinylpyrrolidone, polyglycolides, polyurethanes, alkyl celluloses,cellulose esters, hydroxypropyl derivatives of celluloses and celluloseesters, preformed polymers of poly alkyl acrylates, polyethylene,polystyrene, polyactic acid, polyglycolic acid,poly(lactide-co-glycolide), polycaprolactones, polybutyric acids,polyvaleric acid and copolymers thereof, alginates, chitosans, gelatin,albumin, zein and combinations thereof.
 30. The composition of claim 29,wherein the polymer is poly(lactide-co-glycolide).
 31. The compositionof claim 21, wherein the polymer has a molecular weight in the range of1000 daltons to 150,000 daltons.
 32. The composition of claim 31,wherein the polymer has a molecular weight in the range of 3000 daltonsto 150,000 daltons.
 33. The composition of claim 21, wherein the polymeris present at a concentration of about 0.01% to 30% (w/w).
 34. Thecomposition of claim 33, wherein the polymer is present at aconcentration of about 0.1% to 10% (w/w).